OMES & OMICS in ARTOMES & OMICS in ART

Moncef Benkhalifa, Ph.D. RBMG. Moncef Benkhalifa, Ph.D. RBMG.

Reproductive Biology & GeneticsReproductive Biology & Genetics

Technical & Development DirectorTechnical & Development Director

UNILABS. France UNILABS. France

4. Pré-hybridationHybridation

LavageDécontamination

5. Scan des données – Analyse des résultats

1. Prélèvement de l’échantillonOrganismes / tissues / cellules

2. WGA, RTPCR 3. Amplification (PCR) et marquage fluorescent

The “Omes and Omics”

From James Poscillico

X der(X)

A

D

B

E

X dupXq

21.1

X

21.1

dupXq

25

25

C

q21

q25

0.8 1.2

Tachdjian G, Aboura A, Benkhalifa M et al De novo interstitial direct duplication of Xq21.1q25 associated with skewed X-inactivation pattern.

: Prenatal Diagnosis, October 2005

Custom Profiling: Route to Drug Custom Profiling: Route to Drug Discovery Discovery Application in ovarian cancerApplication in ovarian cancer

Sample 2Sample 2

Chromosome Chromosome segment 8qsegment 8q

Chromosome 13Chromosome 13

Embryon biobsy and single cell MDA

2 kb

600 bp

Lad

der

single cell MDA

6 -8 hours of amplification6 -8 hours of amplification

1;2;3

4;5;6

7;8;9

10;11;12

13;14;15

16;17;18

19;20;21

22;X;Y

Hellani A, Coskun S, Benkhalifa M et al Multiple displacement amplification on single cell and possible PGD applications.

Mol Hum Reprod. 2004 Nov;10(11):847-52.

DNA Multi-Typing DNA Multi-Typing CELL LYSISCELL LYSIS

MULTIPLEX PCR AMPLIFICATION MULTIPLEX PCR AMPLIFICATION

FLUORESCENT SIZING

FLUORESCENT SIZING

AUTOMATED SEQUENCING

AUTOMATED SEQUENCING

ddNTP PRIMER EXTENSION or

“MINISEQUENCING

”

ddNTP PRIMER EXTENSION or

“MINISEQUENCING

”

“NESTED” PCR AMPLIFICATION “NESTED” PCR AMPLIFICATION

MUTATION & FRAGMENT ANALYSIS

MUTATION & FRAGMENT ANALYSIS

Sequencing AnalysisSequencing Analysis MinisequencMinisequencinging

Beta Thalassemia Cod.39 CBeta Thalassemia Cod.39 CTTMethods ComparisonMethods Comparison

NormaNormal l

AlleleAllele

MutateMutated d

AlleleAllele

NormaNormal l

AlleleAllele

MutateMutated d

AlleleAllele

PCR PCR PRODUCT PRODUCT QUANTIFICATIONQUANTIFICATION

NORMAL MALENORMAL MALE KLINEFELTERKLINEFELTER

Peak Peak areasareas

A great number of parameters are detected A great number of parameters are detected simultaneously with microarrayssimultaneously with microarrays

preparation

amplification

reading

interpretation

DNA probes immobilized

RNA or DNA

Microarray for point mutation Analysis: Serial Microarray for point mutation Analysis: Serial Genetics/ATL R&DGenetics/ATL R&D

CFTR microarray: interpretation of results

Universal Microarray PlatformUniversal Microarray Platform

Trisomy 1 Monosomy 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Single Genes

CF Deletion

SNP’s

DNA Fingerprint

Viability Genes

Viable Embryo

Gardner et al

Affymetrix Arrays principle for Affymetrix Arrays principle for transcriptome investigation transcriptome investigation

1,28cm

1 oligonucléotide

1 information

unit

Transcriptomes Analysis

Human Exon Array : ~1,1 .106 d’exons couverts par ~1,8 .106 PSR (Probeset regions).

U133 Plus 2.0 Array : 47,000 transcripts quantified with minimum of 22 séquences (Probeset).

Données contrôlées

ExperimentExperiment

cDNA qualitycontrol

Data Control

Total RNAs extraction quality control

Green colour means downregulated

Red colour means upregulated

What is Proteomics?What is Proteomics?

Proteomics is the large-scale study of Proteomics is the large-scale study of proteins, particularly their structures and proteins, particularly their structures and functions. functions.

Relatively little is known regarding the Relatively little is known regarding the proteome of the human biological cell proteome of the human biological cell systemsystem

Unlike the genome, the proteome is Unlike the genome, the proteome is dynamic constantly changing through its dynamic constantly changing through its biochemical interactions with the genome biochemical interactions with the genome and its response to internal and external and its response to internal and external stimuli stimuli

UptakeUptake ProductionProductionGlucose

Pyruvate

Oxygen

Other Sugars

Lactate

Ammonium

Enzymes, eg LDH

sHLA-G

Other Peptides &

Factors

Amino Acids

µl drop of defined culture medium

HOXA10 regulator

PAF

Amino Acids

[Sakkas and Gardner, Curr. Opin. Obstet. Gynecol. 2005]

CHANGES IN THE CULTURE MEDIA INDICATIVE OF VIABILITY

EMBRYONIC TRANSCRIPTIONmaternal mRNA

GlucosePyruvateLactate

KEY EVENTS IN EMBYRO DEVELOPMENTKEY EVENTS IN EMBYRO DEVELOPMENT

Day 1 Day 3 Day 5

PROTEOMICSPROTEOMICSThe expression of an 8.5-kDa protein The expression of an 8.5-kDa protein biomarker appears to be directly biomarker appears to be directly associated with ongoing human blastocyst associated with ongoing human blastocyst development.development.

[Katz-Jaffe et al. Fertil Steril 2006]

Significant difference in expression

MetabolomicsMetabolomics Metabolomics can be thought of as Metabolomics can be thought of as

the interface between genomics and the interface between genomics and a functional phenotypea functional phenotype

The metabolome represents the final The metabolome represents the final products of gene expressionproducts of gene expression

Assessed by investigating inventory Assessed by investigating inventory of small molecule biomarkers (LMW)of small molecule biomarkers (LMW)

What is Measured?What is Measured?

ClinicallyClinically– How the embryo modifies its How the embryo modifies its

environmentenvironment

BiologicallyBiologically– Changes in concentrations of:Changes in concentrations of:

Constituents•Albumin•Lactate•Pyruvate•Glutamate•Glucose

Functional Groups•CH•NH•OH•SH•C=C

Spent Culture Media Spent Culture Media AnalysesAnalyses

Specific TargetsSpecific Targets(hypotheses-based)(hypotheses-based)

Profile AnalysesProfile Analyses(systems-based)(systems-based)

Utilization of media componentTargeted secretion products

Protein profilingMetabolite profiling

Brison et al ’04;Human Reprod 19:2319

Amino Acid Turnover and ImplantationAmino Acid Turnover and Implantation

No pregnancy Pregnancy

Near Infra-Red Analyses of MediaNear Infra-Red Analyses of Media

Seli et al ’07;Fertil Steril 88:1350-7

Each line is the profile for one embryo

0.6 0.2Step 3: transfer embryo with highest Viability score

Viability Score

Viability Score Methodology

Blank

Step 1: Single embryo culture

Step 2: Spectral analysis of media sample ratioagainst the blank

32

Distribution of the Viability Score of Individual Embryos of the same Morphology: Clinical Trial: MOL BIO-EYLAU

-0.4

-0.2

0 0.2

0.4

0.6

0.8

1

Grade A(very good)

Grade C

Grade D(poor)

Grade B

Morphology

Day 3Day 2

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

B C&D

The impact of the ViaTest score on selectingThe impact of the ViaTest score on selectingembryos of same morphology from embryos of same morphology from

the patient’s cohort: Clinical Trial. MOL BIO-EYLAUthe patient’s cohort: Clinical Trial. MOL BIO-EYLAU

% F

eta

l C

ard

iac

Act

ivit

y P

osi

tive

1

23

4

<0.2070.208 to 0.2880.289 to 0.354

>0.355

Grade A

GenomicsGenomicsProteomicsProteomicsMetabolomicsMetabolomicsEconomicsEconomics

ConclusionsConclusions Developments in genomics will serve to improve Developments in genomics will serve to improve

analysis of entire genome and facilitate genetic analysis of entire genome and facilitate genetic fingerprinting fingerprinting

Analysis of the secretome, plausibly combined with Analysis of the secretome, plausibly combined with carbohydrate utilization and amino acid turnover, and carbohydrate utilization and amino acid turnover, and a general analysis of metabolic footprint, will serve to a general analysis of metabolic footprint, will serve to form the base of new assays of human viability of any form the base of new assays of human viability of any biological process.biological process.

Simplification of proteomics and metabolic analysis Simplification of proteomics and metabolic analysis through microfluidic devices may serve as an economic through microfluidic devices may serve as an economic methodology in clinical practice in the future.methodology in clinical practice in the future.

EylauLaboratory/ EylauLaboratory/ Unilabs.Unilabs.

Paris-Geneva Paris-Geneva

Paul Cohen BacriePaul Cohen BacrieStéphanie BellocStéphanie BellocMartine DumontMartine DumontAnne Marie JuncaAnne Marie JuncaPhilippe RenardPhilippe Renard

Pr Yyves MenezoPr Yyves MenezoPr Alain DalleacPr Alain Dalleac

Gurgan CLINIC. IVF & Genetic

Dept . Ankara. Turkey

Pr T GurganDr A Demirol

T Sari